Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers.

The characterization of the microenvironment of human tumors led to the description of tertiary lymphoid structures (TLS) characterized by mature dendritic cells in a T-cell zone adjacent to B-cell follicle including a germinal center. TLS represent sites of lymphoid neogenesis that develop in most solid cancers. Analysis of the current literature shows that the TLS presence is associated with a favorable clinical outcome for cancer patients, regardless of the approach used to quantify TLS and the stage of the disease. Using several approaches that combine immunohistochemistry, gene expression assays, and flow cytometry on large series of lung tumors, our work demonstrated that TLS are important sites for the initiation and/or maintenance of the local and systemic T- and B-cell responses against tumors. Surrounded by high endothelial venules, they represent a privileged area for the recruitment of lymphocytes into tumors and generation of central-memory T and B cells that circulate and limit cancer progression. TLS can be considered as a novel biomarker to stratify the overall survival risk of untreated cancer patients and as a marker of efficient immunotherapies. The induction and manipulation of cancer-associated TLS using drug agonists and/or biotherapies should open new avenues to treat cancer patients.

Impaired Tumor-Infiltrating T Cells in Patients with Chronic Obstructive Pulmonary Disease Impact Lung Cancer Response to PD-1 Blockade.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) have a higher prevalence of lung cancer. The chronic inflammation associated with COPD probably promotes the earliest stages of carcinogenesis. However, once tumors have progressed to malignancy, the impact of COPD on the tumor immune microenvironment remains poorly defined, and its effects on immune-checkpoint blockers' efficacy are still unknown. OBJECTIVES: To study the impact of COPD on the immune contexture of non-small cell lung cancer. METHODS: We performed in-depth immune profiling of lung tumors by immunohistochemistry and we determined its impact on patient survival (n = 435). Tumor-infiltrating T lymphocyte (TIL) exhaustion by flow cytometry (n = 50) was also investigated. The effectiveness of an anti-PD-1 (programmed cell death-1) treatment (nivolumab) was evaluated in 39 patients with advanced-stage non-small cell lung cancer. All data were analyzed according to patient COPD status. MEASUREMENTS AND MAIN RESULTS: Remarkably, COPD severity is positively correlated with the coexpression of PD-1/TIM-3 (T-cell immunoglobulin and mucin domain-containing molecule-3) by CD8 T cells. In agreement, we observed a loss of CD8 T cell-associated favorable clinical outcome in COPD+ patients. Interestingly, a negative prognostic value of PD-L1 (programmed cell death ligand 1) expression by tumor cells was observed only in highly CD8 T cell-infiltrated tumors of COPD+ patients. Finally, data obtained on 39 patients with advanced-stage non-small cell lung cancer treated by an anti-PD-1 antibody showed longer progression-free survival in COPD+ patients, and also that the association between the severity of smoking and the response to nivolumab was preferentially observed in COPD+ patients. CONCLUSIONS: COPD is associated with an increased sensitivity of CD8 tumor-infiltrating T lymphocytes to immune escape mechanisms developed by tumors, thus suggesting a higher sensitivity to PD-1 blockade in patients with COPD.

Intratumoral Immune Cell Densities Are Associated with Lung Adenocarcinoma Gene Alterations.

RATIONALE: Tumor-infiltrating immune cells affect lung cancer outcome. However, the factors that influence the composition and function of the tumor immune environment remain poorly defined and need investigation, particularly in the era of immunotherapy. OBJECTIVES: To determine whether the tumoral immune environment is related to lung adenocarcinoma mutations. METHODS: This retrospective cohort included 316 consecutive patients with lung adenocarcinoma (225 men; 258 smokers) studied from 2001 to 2005 in a single center. We investigated the association of densities of intratumoral mature dendritic cells (mDCs), CD8+ T cells, neutrophils, and macrophages with clinical and pathological variables and tumor cell mutation profiles obtained by next-generation sequencing. MEASUREMENTS AND MAIN RESULTS: In 282 tumors, we found 460 mutations, mainly in TP53 (59%), KRAS (40%), STK11 (24%), and EGFR (14%). Intratumoral CD8+ T-cell density was high in smokers (P = 0.02) and TP53-mutated tumors (P = 0.02) and low in BRAF-mutated tumors (P = 0.005). Intratumoral mDC density was high with low pathological tumor stage (P = 0.01) and low with STK11 mutation (P = 0.004). Intratumoral neutrophil density was high and low with BRAF mutation (P = 0.04) and EGFR mutation (P = 0.02), respectively. Intratumoral macrophage density was low with EGFR mutation (P = 0.01). Intratumoral CD8+ T-cell and mDC densities remained strong independent markers of overall survival (P = 0.001 and P = 0.02, respectively). CONCLUSIONS: Intratumoral immune cell densities (mDCs, CD8+ T cells, neutrophils, macrophages) were significantly associated with molecular alterations in adenocarcinoma underlying the interactions between cancer cells and their microenvironment.

Presence of B cells in tertiary lymphoid structures is associated with a protective immunity in patients with lung cancer.

RATIONALE: It is now well established that immune responses can take place outside of primary and secondary lymphoid organs. We previously described the presence of tertiary lymphoid structures (TLS) in patients with non-small cell lung cancer (NSCLC) characterized by clusters of mature dendritic cells (DCs) and T cells surrounded by B-cell follicles. We demonstrated that the density of these mature DCs was associated with favorable clinical outcome. OBJECTIVES: To study the role of follicular B cells in TLS and the potential link with a local humoral immune response in patients with NSCLC. METHODS: The cellular composition of TLS was investigated by immunohistochemistry. Characterization of B-cell subsets was performed by flow cytometry. A retrospective study was conducted in two independent cohorts of patients. Antibody specificity was analyzed by ELISA. MEASUREMENTS AND MAIN RESULTS: Consistent with TLS organization, all stages of B-cell differentiation were detectable in most tumors. Germinal center somatic hypermutation and class switch recombination machineries were activated, associated with the generation of plasma cells. Approximately half of the patients showed antibody reactivity against up to 7 out of the 33 tumor antigens tested. A high density of follicular B cells correlated with long-term survival, both in patients with early-stage NSCLC and with advanced-stage NSCLC treated with chemotherapy. The combination of follicular B cell and mature DC densities allowed the identification of patients with the best clinical outcome. CONCLUSIONS: B-cell density represents a new prognostic biomarker for NSCLC patient survival, and makes the link between TLS and a protective B cell-mediated immunity.

Shaping of an effective immune microenvironment to and by cancer cells.

A high density of intratumoral effector memory CD8+/Th1 T cells is associated with favorable prognosis in most cancers and may be induced or increased by immunotherapy. Efficient adaptive immune reactions are shaped in tumor adjacent tertiary lymphoid structures, which exhibit all characteristics of immunity generating lymphoid formations in reactive lymph nodes. Malignant tumor cells impact favorably or deleteriously their immune microenvironment if they bear genetic mutations that result in neo-antigens or by producing chemokines and cytokines that recruit lymphocytes and myeloid cells or increase inflammation and neo-angiogenesis. This intricate network of interactions results in control or escape of tumors, and its understanding will help define goals to monitor efficiency of immunotherapies.

Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-γ contributes to modulate immune responses.

CD161 is a C-type lectin-like receptor expressed on human natural killer (NK) cells and subsets of T cells. CD161 has been described as an inhibitory receptor that regulates NK cell-mediated cytotoxicity and IFN-γ production. Its role on T cells has remained unclear. Studies have shown that triggering of CD161 enhances NK T cell proliferation and T cell-IFN-γ production while inhibiting TNF-α production by CD8(+) T cells. Lectin-like transcript 1 (LLT1), the ligand of CD161, was found to be expressed on Toll-like receptor (TLR)-activated plasmacytoid and monocyte-derived dendritic cells (DC) and on activated B cells. Using newly developed anti-LLT1 mAbs, we show that LLT1 is not expressed on the surface of circulating B and T lymphocytes, NK cells, monocytes, and dendritic cells but that LLT1 is up-regulated upon activation. Not only TLR-stimulated dendritic cells and B cells but also T cell receptor-activated T cells and activated NK cells up-regulate LLT1. Interestingly, IFN-γ increases LLT1 expression level on antigen-presenting cells. LLT1 is also induced on B cells upon viral infection such as Epstein-Barr virus or HIV infection and in inflamed tonsils. Finally, expression of LLT1 on B cells inhibits NK cell function but costimulates T cell proliferation or IFN-γ production, and coengagement of CD161 with CD3 increases IL-17 secretion. Altogether, our results point toward a role for LLT1/CD161 in modulating immune responses to pathogens.

Mechanisms of NK cell activation: CD4(+) T cells enter the scene.

Natural killer (NK) cells are innate lymphocytes involved in immunosurveillance through their cytotoxic activity and their capacity to secrete inflammatory cytokines. NK cell activation is necessary to initiate effector functions and results from a complex series of molecular and cellular events. We review here the signals that trigger NK cells and discuss recent findings showing that, besides antigen-presenting cells, T cells can play a central role in the initiation of NK cell activation in lymph nodes.

LLT1-CD161 Interaction in Cancer: Promises and Challenges.

The success of immune checkpoint therapy in cancer has changed our way of thinking, promoting the design of future cancer treatments that places the immune system at the center stage. The knowledge gained on immune regulation and tolerance helped the identification of promising new clinical immune targets. Among them, the lectin-like transcript 1 (LLT1) is the ligand of CD161 (NKR-P1A) receptor expressed on natural killer cells and T cells. LLT1/CD161 interaction modulates immune responses but the exact nature of the signals delivered is still partially resolved. Investigation on the role of LLT1/CD161 interaction has been hampered by the lack of functional homologues in animal models. Also, some studies have been misled by the use of non-specific reagents. Recent studies and meta-analyses of single cell data are bringing new insights into the function of LLT1 and CD161 in human pathology and notably in cancer. The advances made on the characterization of the tumor microenvironment prompt us to integrate LLT1/CD161 interaction into the equation. This review recapitulates the key findings on the expression profile of LLT1 and CD161, their regulation, the role of their interaction in cancer development, and the relevance of targeting LLT1/CD161 interaction.

Regulatory T cells infiltrate the tumor-induced tertiary lymphoïd structures and are associated with poor clinical outcome in NSCLC.

On one hand, regulatory T cells (Tregs) play an immunosuppressive activity in most solid tumors but not all. On the other hand, the organization of tumor-infiltrating immune cells into tertiary lymphoid structures (TLS) is associated with long-term survival in most cancers. Here, we investigated the role of Tregs in the context of Non-Small Cell Lung Cancer (NSCLC)-associated TLS. We observed that Tregs show a similar immune profile in TLS and non-TLS areas. Autologous tumor-infiltrating Tregs inhibit the proliferation and cytokine secretion of CD4+ conventional T cells, a capacity which is recovered by antibodies against Cytotoxic T-Lymphocyte-Associated protein-4 (CTLA-4) and Glucocorticoid-Induced TNFR-Related protein (GITR) but not against other immune checkpoint (ICP) molecules. Tregs in the whole tumor, including in TLS, are associated with a poor outcome of NSCLC patients, and combination with TLS-dendritic cells (DCs) and CD8+ T cells allows higher overall survival discrimination. Thus, Targeting Tregs especially in TLS may represent a major challenge in order to boost anti-tumor immune responses initiated in TLS.

Characterization of alternatively spliced transcript variants of CLEC2D gene.

Lectin-like transcript 1 (LLT1) encoded by CLEC2D gene is a C-type lectin-like molecule interacting with human CD161 (NKR-P1A) receptor expressed by natural killer cells and subsets of T cells. Using RT-PCR and sequencing, we identified several CLEC2D alternatively spliced transcript variants generated by exon skipping. In addition to the reported transcript variants 1 (LLT1) and 2, we identified a novel splice variant 4 and transcripts coding for putative soluble proteins. CLEC2D transcripts were detected primarily in hematopoietic cell lines and were found to be co-induced by the same activation signals. Although very low amounts of putative soluble CLEC2D protein isoforms could be produced by transfectants, CLEC2D isoforms 2 and 4 were efficiently expressed. By contrast to LLT1, which was detected on the cell surface, isoform 2 and 4 remained in the endoplasmic reticulum where they formed homodimers or heterodimers with LLT1. They failed to interact with CD161, leaving LLT1 as the sole ligand for this receptor. CLEC2D therefore uses gene splicing to generate protein isoforms that are structurally distinct and that have different biological activities.

Tertiary Lymphoid Structure-B Cells Narrow Regulatory T Cells Impact in Lung Cancer Patients.

The presence of tertiary lymphoid structures (TLS) in the tumor microenvironment is associated with better clinical outcome in many cancers. In non-small cell lung cancer (NSCLC), we have previously showed that a high density of B cells within TLS (TLS-B cells) is positively correlated with tumor antigen-specific antibody responses and increased intratumor CD4+ T cell clonality. Here, we investigated the relationship between the presence of TLS-B cells and CD4+ T cell profile in NSCLC patients. The expression of immune-related genes and proteins on B cells and CD4+ T cells was analyzed according to their relationship to TLS-B density in a prospective cohort of 56 NSCLC patients. We observed that tumor-infiltrating T cells showed marked differences according to TLS-B cell presence, with higher percentages of naïve, central-memory, and activated CD4+ T cells and lower percentages of both immune checkpoint (ICP)-expressing CD4+ T cells and regulatory T cells (Tregs) in the TLS-Bhigh tumors. A retrospective study of 538 untreated NSCLC patients showed that high TLS-B cell density was even able to counterbalance the deleterious impact of high Treg density on patient survival, and that TLS-Bhigh Treglow patients had the best clinical outcomes. Overall, the correlation between the density of TLS-Bhigh tumors with early differentiated, activated and non-regulatory CD4+ T cell cells suggest that B cells may play a central role in determining protective T cell responses in NSCLC patients.

Non-destructive optical monitoring of grape maturation by proximal sensing.

A new, commercial, fluorescence-based optical sensor for plant constituent assessment was recently introduced. This sensor, called the Multiplex(®) (FORCE-A, Orsay, France), was used to monitor grape maturation by specifically monitoring anthocyanin accumulation. We derived the empirical anthocyanin content calibration curves for Champagne red grape cultivars, and we also propose a general model for the influence of the proportion of red berries, skin anthocyanin content and berry size on Multiplex(®) indices. The Multiplex(®) was used on both berry samples in the laboratory and on intact clusters in the vineyard. We found that the inverted and log-transformed far-red fluorescence signal called the FERARI index, although sensitive to sample size and distance, is potentially the most widely applicable. The more robust indices, based on chlorophyll fluorescence excitation ratios, showed three ranges of dependence on anthocyanin content. We found that up to 0.16 mg cm(-2), equivalent to approximately 0.6 mg g(-1), all indices increase with accumulation of skin anthocyanin content. Excitation ratio-based indices decrease with anthocyanin accumulation beyond 0.27 mg cm(-2). We showed that the Multiplex(®) can be advantageously used in vineyards on intact clusters for the non-destructive assessment of anthocyanin content of vine blocks and can now be tested on other fruits and vegetables based on the same model.

Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions.

BACKGROUND: Natural killer (NK) cells play a crucial role in tumor immunosurveillance through their cytotoxic effector functions and their capacity to interact with other immune cells to build a coordinated antitumor immune response. Emerging data reveal NK cell dysfunction within the tumor microenvironment (TME) through checkpoint inhibitory molecules associated with a regulatory phenotype. OBJECTIVE: We aimed at analyzing the gene expression profile of intratumoral NK cells compared with non-tumorous NK cells, and to characterize their inhibitory function in the TME. METHODS: NK cells were sorted from human lung tumor tissue and compared with non- tumoral distant lungs. RESULTS: In the current study, we identify a unique gene signature of NK cell dysfunction in human non-small cell lung carcinoma (NSCLC). First, transcriptomic analysis reveals significant changes related to migratory pattern with a downregulation of sphingosine-1-phosphate receptor 1 (S1PR1) and CX3C chemokine receptor 1 (CX3CR1) and overexpression of C-X-C chemokine receptor type 5 (CXCR5) and C-X-C chemokine receptor type 6 (CXCR6). Second, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and killer cell lectin like receptor (KLRC1) inhibitory molecules were increased in intratumoral NK cells, and CTLA-4 blockade could partially restore MHC class II level on dendritic cell (DC) that was impaired during the DCs/NK cell cross talk. Finally, NK cell density impacts the positive prognostic value of CD8+ T cells in NSCLC. CONCLUSIONS: These findings demonstrate novel molecular cues associated with NK cell inhibitory functions in NSCLC.

Identification of Tertiary Lymphoid Structure-Associated Follicular Helper T Cells in Human Tumors and Tissues.

Follicular helper T (Tfh) cells are major components of the humoral immune response due to their pivotal role in germinal center formation and antibody affinity maturation following B-cell isotype switching. This CD4+ T-cell subtype is mainly found in the B-cell zone of secondary lymphoid organs as well as in tertiary lymphoid structures (TLS), which are highly organized structures composed of T and B cells, occasionally found at the invasive margin in the tumor microenvironment.We describe here how to perform immunofluorescence staining of tumor tissue sections and multicolor flow cytometry on tumor cell suspensions to identify and visualize these TLS-associated Tfh cells within the tumor microenvironment of various human cancers. These assays take advantage of combinations of markers and molecules involved in Tfh differentiation and function.

Development of Tools for the Selective Visualization and Quantification of TLS-Immune Cells on Tissue Sections.

Tertiary lymphoid structures (TLS) are considered as genuine markers of inflammation. Their presence within inflamed tissues or within the tumor microenvironment has been associated with the local development of an active immune response. While high densities of TLS are correlated with disease severity in autoimmune diseases or during graft rejection, it has been associated with longer patient survival in many cancer types. Their efficient visualization and quantification within human tissues may represent new tools for helping clinicians in adjusting their therapeutic strategy. Some immunohistochemistry (IHC) protocols are already used in the clinic to appreciate the level of immune infiltration in formalin-fixed, paraffin-embedded (FFPE) tissues. However, the use of two or more markers may sometimes be useful to better characterize this immune infiltrate, especially in the case of TLS. Besides the growing development of multiplex labeling approaches, imaging can also be used to overcome some technical difficulties encountered during the immunolabeling of tissues with several markers.This chapter describes IHC methods to visualize in a human tissue (tumoral or not) the presence of TLS. These methods are based on the immunostaining of four TLS-associated immune cell populations, namely follicular B cells, follicular dendritic cells (FDCs), mature dendritic cells (mDCs), and follicular helper T cells (TFH), together with non-TFH T cells. Methodologies for subsequent quantification of TLS density are also proposed, as well as a virtual multiplexing method based on image registration using the open-source software ImageJ (IJ), aiming at co-localizing several immune cell populations from different IHC stainings performed on serial tissue sections.

Expression of LLT1 and its receptor CD161 in lung cancer is associated with better clinical outcome.

Co-stimulatory and inhibitory receptors expressed by immune cells in the tumor microenvironment modulate the immune response and cancer progression. Their expression and regulation are still not fully characterized and a better understanding of these mechanisms is needed to improve current immunotherapies. Our previous work has identified a novel ligand/receptor pair, LLT1/CD161, that modulates immune responses. Here, we extensively characterize its expression in non-small cell lung cancer (NSCLC). We show that LLT1 expression is restricted to germinal center (GC) B cells within tertiary lymphoid structures (TLS), representing a new hallmark of the presence of active TLS in the tumor microenvironment. CD161-expressing immune cells are found at the vicinity of these structures, with a global enrichment of NSCLC tumors in CD161+ CD4+ and CD8+ T cells as compared to normal distant lung and peripheral blood. CD161+ CD4+ T cells are more activated and produce Th1-cytokines at a higher frequency than their matched CD161-negative counterparts. Interestingly, CD161+ CD4+ T cells highly express OX40 co-stimulatory receptor, less frequently 4-1BB, and display an activated but not completely exhausted PD-1-positive Tim-3-negative phenotype. Finally, a meta-analysis revealed a positive association of CLEC2D (coding for LLT1) and KLRB1 (coding for CD161) gene expression with favorable outcome in NSCLC, independently of the size of T and B cell infiltrates. These data are consistent with a positive impact of LLT1/CD161 on NSCLC patient survival, and make CD161-expressing CD4+ T cells ideal candidates for efficient anti-tumor recall responses.

TP53, STK11, and EGFR Mutations Predict Tumor Immune Profile and the Response to Anti-PD-1 in Lung Adenocarcinoma.

Purpose: By unlocking antitumor immunity, antibodies targeting programmed cell death 1 (PD-1) exhibit impressive clinical results in non-small cell lung cancer, underlining the strong interactions between tumor and immune cells. However, factors that can robustly predict long-lasting responses are still needed.Experimental Design: We performed in-depth immune profiling of lung adenocarcinoma using an integrative analysis based on immunohistochemistry, flow-cytometry, and transcriptomic data. Tumor mutational status was investigated using next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort according to tumor mutational profiles and PD-L1 expression, and a public clinical database was used to validate the results obtained.Results: We showed that distinct combinations of STK11, EGFR, and TP53 mutations were major determinants of the tumor immune profile (TIP) and of the expression of PD-L1 by malignant cells. Indeed, the presence of TP53 mutations without co-occurring STK11 or EGFR alterations (TP53-mut/STK11-EGFR-WT), independently of KRAS mutations, identified the group of tumors with the highest CD8 T-cell density and PD-L1 expression. In this tumor subtype, pathways related to T-cell chemotaxis, immune cell cytotoxicity, and antigen processing were upregulated. Finally, a prolonged progression-free survival (PFS: HR = 0.32; 95% CI, 0.16-0.63, P < 0.001) was observed in anti-PD-1-treated patients harboring TP53-mut/STK11-EGFR-WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression.Conclusions: Our study reveals that different combinations of TP53, EGFR, and STK11 mutations, together with PD-L1 expression by tumor cells, represent robust parameters to identify best responders to PD-1 blockade. Clin Cancer Res; 24(22); 5710-23. ©2018 AACR.

Tumor-Infiltrating and Peripheral Blood T-cell Immunophenotypes Predict Early Relapse in Localized Clear Cell Renal Cell Carcinoma.

Purpose: The efficacy of PD-1 checkpoint blockade as adjuvant therapy in localized clear cell renal cell carcinoma (ccRCC) is currently unknown. The identification of tumor microenvironment (TME) prognostic biomarkers in this setting may help define which patients could benefit from checkpoint blockade and uncover new therapeutic targets.Experimental Design: We performed multiparametric flow cytometric immunophenotypic analysis of T cells isolated from tumor tissue [tumor-infiltrating lymphocytes (TIL)], adjacent non-malignant renal tissue [renal-infiltrating lymphocytes (RIL)], and peripheral blood lymphocytes (PBL), in a cohort of patients (n = 40) with localized ccRCC. Immunophenotypic data were integrated with prognostic and histopathologic variables, T-cell receptor (TCR) repertoire analysis of sorted CD8+PD-1+ TILs, tumor mRNA expression, and digital quantitative immunohistochemistry.Results: On the basis of TIL phenotypic characterization, we identified three dominant immune profiles in localized ccRCC: (i) immune-regulated, characterized by polyclonal/poorly cytotoxic CD8+PD-1+Tim-3+Lag-3+ TILs and CD4+ICOS+ cells with a Treg phenotype (CD25+CD127-Foxp3+/Helios+GITR+), that developed in inflamed tumors with prominent infiltrations by dysfunctional dendritic cells and high PD-L1 expression; (ii) immune-activated, enriched in oligoclonal/cytotoxic CD8+PD-1+Tim-3+ TILs, that represented 22% of the tumors; and (iii) immune-silent, enriched in TILs exhibiting RIL-like phenotype, that represented 56% of patients in the cohort. Only immune-regulated tumors displayed aggressive histologic features, high risk of disease progression in the year following nephrectomy, and a CD8+PD-1+Tim-3+ and CD4+ICOS+ PBL phenotypic signature.Conclusions: In localized ccRCC, the infiltration with CD8+PD-1+Tim-3+Lag-3+ exhausted TILs and ICOS+ Treg identifies the patients with deleterious prognosis who could benefit from adjuvant therapy with TME-modulating agents and checkpoint blockade. This work also provides PBL phenotypic markers that could allow their identification. Clin Cancer Res; 23(15); 4416-28. ©2017 AACR.

MHC class I-related chain A conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells.

PURPOSE: As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I-related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. EXPERIMENTAL DESIGN: Recombinant MICA (rMICA) was chemically conjugated to Fab' fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. RESULTS: Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell-mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. CONCLUSIONS: These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.